RA-42, a polypeptide with 42 amino acids, has previously been shown to react with p-nitrophenyl fumarate (1) and site selectively form an amide at the side chain of ornithine-15 in aqueous solution and in 10 vol% 2,2,2-trifluoroethanol (TFE). In order to investigate the role of the medium on structure and on reactivity the mean residue ellipticity at 222 nm has been measured by circular dichroism spectroscopy as a function of TFE concentration and the second-order rate constant for the reaction between RA-42 and 1 has been measured in 30 vol% TFE. The correlation was poor between the mean residue ellipticity and the second-order rate constants, and the effect of increasing the TFE concentration was therefore most likely to increase the strength of electrostatic interactions in the transition state due to the decreased polarity of the solvent. The ratio of second-order rate constants between that of RA-42 and that of 4-methylimidazole (4-MeIm), k2(RA-42)/k2(4-MeIm), increased with TFE concentration suggesting that there was little contribution to transition state binding in helix II since there is little hairpin conformation left in 30 vol% TFE. Finally, the effect on structure of the introduced ornithines was determined by measuring the free energy of unfolding. Although the helix–loop–helix motif was destabilised by the incorporation of positively charged residues, the effect on reactivity was small, suggesting that polypeptides are useful as templates in the rational design of catalysts.